The generation of libraries of small molecules and selection of those molecules that bind uniquely to a target of interest is important for drug discovery. The production of genetically-encoded libraries, in which each library member is linked to an information template, such as DNA or RNA, makes it possible to process large chemical libraries without separating individual library members into individual solutions and reaction vessels. One can select target molecules from mixtures of genetically-encoded molecules and identify or amplify the selected molecule of interest using its information template.
Phage display is one example of a genetically-encoded library. (Scott et al., 1990). Phage display is a well known technique used in the analysis, display and production of protein antigens, especially human proteins of interest. Phage display is a process during which the phage, a bacterial virus, is made to expose or “display” different peptides or proteins including human antibodies on its surface. Through genetic engineering, peptides or proteins of interest are attached individually to a phage cell surface protein molecule (usually Gene III protein, g3p). In such a phage population (phage library), each phage carries a gene for a different peptide or protein—g3p fusion and exposes it on its surface. Through a variety of selection procedures, phages that “display” binders to specific target molecules of interest can be identified and isolated. These binders can include interaction partners of a protein to determine new functions or mechanisms of function of that protein, peptides that recognize and bind to antigens (for use in diagnosis and therapeutic targeting, for example), and proteins involved in protein-DNA interactions (for example, novel transcription factors).
The phage display technique can be very useful in discovery and development of pharmaceutical andlor diagnostic products. In phage display the entire phage binds and can be eluted from an immobilized target molecule. Since the phage remains infective it can inject its DNA into bacterial cells and is amplified. The main limitation of phage display, however, is the occurrence of non-specific adsorption of phages during the binding stage, which necessitates enrichment over several rounds and individually tailored washing and elution conditions. Phage display methods are usually restricted to the production of libraries, which can be encoded by direct DNA-RNA-protein information transfer. These methods are typically limited to linear sequences of peptides, made of only 20 natural amino acids.
RNA and ribosome display are other techniques known in the art that permit display of naturally-made peptides on information templates. The amplification of libraries of peptides attached to RNA requires an in vitro translation system to generate or reamplify the library. The generation and use of such translation systems can be expensive and time consuming. The use of self-replicating species such as phage, yeast, or bacteria simplifies amplification of libraries because each library member is amplified “spontaneously”, when given the appropriate resources. For example, for phage displayed libraries, adding one phage to a simple culture broth with bacteria can produce an arbitrarily large population of phage for a very low cost.
Genetic encoding of small molecules has been proposed in the 1990s (Brenner et al., 1990) and several implementations of such encoding strategies have been developed by different groups, including Lerner and Janda (Scripps), Lu (Harvard) and Harbury (Stanford). The encoding of small-molecules developed by these groups, however, is significantly more complex than technologies for display of polypeptides. Further, encoding strategies can be extremely difficult in terms of synthesis and achieving rapid round-to-round iterations in the process.
Another example of small-molecule display technology is through the use of encoded display of molecules derived from peptides via enzymatic or chemical post-translational modifications (cPTM). Typically, these methods use organic synthesis on the peptides to make peptide derivatives. Unlike the display of arbitrary organic molecules, the display of peptide-derived molecules is generally simpler because it builds on readily-available genetically-encoded peptide libraries.
It is known that an entire peptide library can be modified by uniform chemical modification. Selection from the modified library and sequencing of the DNA yields peptide sequences from which the modified peptide derivatives can be made. Several methods exist which involve conversion of libraries of phage-displayed polypeptides to libraries of peptide derivatives.
US Patent Publication 2010/0317547 to Winter and Heinis, describes specific modifications of phage displayed by alkylation of cysteine residues.
US Patent Publication 2009/0137424 to Schultz et al, describes specific modifications of phage-displayed peptide libraries by dipolar cycloaddition on azido phenyl alanine (AzPhe).
Bulk biochemical methods, such as western blot and mass spectrometry, are often used, to quantify the amount of product obtained or to determine the success of generating the desired reaction products. In the absence of this characterization, the synthesis cannot be claimed to be reliable or reproducible. Reactions used for synthesis of such libraries of peptide derivatives have typically been validated using one phage clone or one purified peptide. The actual synthesis of libraries is typically done without characterization under conditions optimized for a peptide but the efficiency of such synthesis is unknown. The quality of the libraries generated by this method is, thus, usually unknown. While selection from these libraries can provide useful non-peptidic molecules, overall the efficiency of such selection is unclear. The characterization and improvement of reaction is important for developing new chemically-modified libraries
US Patent Publication 2013/050083 to Derda et al, describes a method for quantification of such modifications and selection of new strategies for effective modification.
It has been shown that cPTM of mRNA- or phage-displayed peptides can have similar advantages as mRNAiphage display while allowing the selection of ligands that cannot be encoded by conventional ribosomal synthesis. Selection of cPTM-libraries is a rapidly growing method that has been used by lead discovery academic research groups, start-up companies (Peptidream, Ra Pharma, Bicycle therapeutics) and large pharmaceutical companies (e.g., Pfizer5). In drug discovery, the display of cPTM-peptides facilitates developing a class of drugs that combines the advantages of “small-molecule” and “biological”-classes of drugs. Due to their small size, peptide-derivatives can have tissue permeability akin to that of small molecules, while genetic selection allows for rapid discovery and optimization of these molecules.
One of the problems with cPTM-libraries is genetic encoding of the modifications. While it is possible to convert linear peptide libraries to cyclic (Kawakami et al., 2013; Josephson et al., 2005; Jafari et al., 2014), bicyclic (Heinis et al., 2009), or glycosylated (Ng et al., 2012) molecules, multiple modifications cannot be performed on the same library because the identity of modification cannot be traced.
The screening of identical libraries with different modifications is known but it is typically done by parallel modification of different libraries, their parallel panning and processing, and their parallel sequencing (Chen et al., 2014; Schlippe et al., 2012). If two or more chemical modifications are combined into one library, such mixed library is difficult to analyze because it is usually not possible to distinguish between modified phage particles. There is no information about modification on the genetic level and there are no obvious and universal strategies for encoding or decoding such modifications.
There exists a need to provide an effective method of identifying molecules for drug discovery.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.